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Cloning
Add a poly-A tail to blunt-ended samples or old samples Some DNA polymerases with proofreading activity generate blunt-ended fragments. Furthermore, the poly-A tail generated during PCR degrades rapidly, so fragments that are any more than a day or two old are also generally blunt ended. A poly-A tail is required for successful ligation into the pGEM-T vector. Old samples and those generated using a DNA polymerase with proofreading activity must be modified using the following A-tailing procedure: #Start with 1-7ul of purified, blunt-ended PCR fragment. #Add 1ul Taq DNA Polymerase 10X Reaction Buffer with MgCl2. #Add dATP to a final concentration of 0.2mM (0.2ul dNTP mix). #Add 1ul Taq ''DNA Polymerase (5 U/ul). #Add deionized water to a final reaction volume of 10ul. #Incubate at 70°C for 15-30 minutes. #Use 1-2ul in a ligation reaction with the pGEM-T or pGEM-T Easy Vector. Ligation Protocol #Briefly centrifuge the pGEM-T Vector and control insert DNA tubes to collect contents. #Set up ligation reactions as described below, vortexing 2X Rapid Ligation Buffer vigorously before use: #Mix the reactions by pipetting. Incubate for 1 hour at room temperature. Alternatively, if the maximum number of transformants is required, incubate the reactions overnight (16-24 hours) at 4°C. Optimizing Insert:Vector Molar Ratios A 1:1 molar ratio of PCR fragment insert to vector is optimal. However, ratios of 3:1 - 1:3 are generally good, and ratios as extreme as 8:1 - 1:8 have been used successfully. The concentration of PCR product should be determined using photospectometry. To calculate the appropriate amound of PCR product (insert) to include in the ligation reaction, use the following equation: For example, how much 0.5kb PCR product should be added to a ligation in which 50ng of 3.0kb vector will be used if a 3:1 insert:vector molar ratio is desired? Using the same parameters for a 1:1 insert:vector molar ratio, 8.3ng of a 0.5kb insert would be required Promega's biomath calculator can be used to determine the amount of insert DNA needed. The pGEM-T Vector size is 3000bp and the pGEM-T Easy Vector size is 3015bp. Transformation Protocol Use high-efficiency competent cells (>1 X 108cfu / ug DNA) for transformations. #Prepare two LB/ampicillin/IPTG/X-Gal plates for each ligation reaction, plus two extra for determining transformation efficiency. Equilibrate to room temperature. Set water bath to 42°C. #Centrifuge ligation tubes. Add 2ul of each ligation reaction to a 1.5 ml microcentrifuge tube on ice. Set up another tube on ice with 0.1 ng uncut plasmid for determination of transformation efficiency. #Quickly thaw competent cells by warming between hands and immediately dispense 50ul of cells into each of the tubes prepared in step 2 (use 100ul of cells for determining transformation efficiency). #Gently flick the tubes to mix and place them on ice for 20 minutes. #Heat shock cells for 45-55 seconds in a water bath at exactly 42°C. #Immediately return to ice for 2 minutes. #Add 950ul LB medium to each tube (900ul to the tube containing cells transformed with uncut plasmid. #Incubate for 1 hour at 37°C with shaking at 150 rpm. #Plate 100ul of each transformation culture onto duplicate LB/ampicillin/IPTG/X-Gal plates. For determining the transformation efficiency, a 1:10 dilution with LB medium should be used for plating. #Incubate plates overnight (16-24 hours) at 37°C. Plates should be incubated upsidedown to prevent condensation from dripping onto growing bacterial colonies. Determining Transformation Efficiency After 100ul of competent cells are transformed with 0.1ng uncut plasmid DNA, the transformation reaction is added to 900ul of LB medium (0.1ng DNA/ml). From the volume, a 1:10 dilution with LB medium is made (0.01ng DNA/ml) and 100ul is plated on duplicate plates (0.001ng DNA/100ul). If 200 colonies are obtained (average of the two plates), what is the transformation efficiency? Screening Transformants for Inserts Successful cloning of an insert into the pGEM-T or pGEM-T Easy Vector interrupts the coding sequence of B-galactosidease. Clones containing PCR products produce white colonies, whereas those that contain non-ligated plasmid produce blue colonies as a result of B-galactosidase activity. Occasionally, blue colonies result from PCR fragments that are cloned in-frame with the ''lacZ gene. These fragments are usually a multiple of 3 base pairs long and do not contain in-frame stop codons. For each transformation reaction, select at least two transformant colonies for incubation and plasmid purification. Select white colonies that have no touching neighbors for incubation. This will ensure the homogeneity of genetic material in the incubated colony. Incubating Selected Colonies #Add 2ml LB medium and 2ul ampicillin (100mg/ml) to two sterile 13ml sarstead tubes per plate. #For each plate, pick two white colonies that are isolated from their neighbors. Gently scrape one colony with a sterile pipet tip to transfer some cells onto the tip, and dispense into one of the tubes prepared in step 1. #Repeat for a second colony. Depending on the purity of your starting PCR prduct, and the efficiency of the transformation reaction, it may be necessary to screen more colonies to identify clones that contain the PCR product of interest. #Incubate the tubes overnight (16-24 hours) at 37°C with shaking at 150-250 rpm. Once the tubes have been incubated, the medium should be cloudy as a result of cellular replication. #Cultures can be used immediately or stored for up to a week at 4°C before use in plasmid purification. Category:Lab techniques